Molecular cloning, characterization and expression analysis of cathepsin D gene from turbot Scophthalmus maximus.
Cathepsin D is a lysosomal endoproteolytic aspartic proteinase which also has been found in endosomes of macrophage. It is thought to play key roles in the developmental and physiological process of animals. The EST sequence of turbot (Scophthalmus maximus L.) cathepsin D was obtained from a subtractive cDNA library. In the present study, 5'-RACE and 3'-RACE were carried out to obtain the complete cDNA sequence of turbot cathepsin D, which contained a 91 bp 5'-UTR, a 1191 bp open reading frame encoding 396 amino acids, and a 329 bp 3'-UTR. The deduced amino acid sequence of the cathepsin D consisted of a signal peptide of 18 aa, a leader peptide extending 43 aa, and a mature peptide of 335 aa. BLAST analysis revealed that turbot cathepsin D shared high similarity with other known cathepsin D, and it showed significant homology with that of Barramundi (Lates calcarifer B., 89% aa similarity). Quantitative real-time PCR (q PCR) demonstrated that the highest expression level of the turbot cathepsin D was in liver. After turbot were challenged with Vibrio harveyi, the lowest expression levels of cathepsin D in liver, spleen and head kidney were detected at 8 h. This result was different from the expression of MHCII of which the expression lever was increased upon challenge. The expression levels of cathepsin D in liver and head kidney increased gradually after 8 h and exceeded the background level after 24 h. In spleen, the expression level was reinforced after 8 h and kept at level that was higher than the original level after 12 h. The results suggested that cathepsin D might process antigens for presentation to the immune system and have synergetic effect with apoptosis pathway until 12 h after injection.
Jia
Airong
A
Department of Marine Biology, Ocean University of China, Qingdao 266003, China.
Zhang
Xiao-Hua
XH
D000595:Amino Acid Sequence; D000818:Animals; D002402:Cathepsin D / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D005393:Fish Diseases / Q000276:immunology / Q000382:microbiology; D005412:Flatfishes / Q000145:classification / Q000235:genetics* / Q000276:immunology*; D005786:Gene Expression Regulation; D008221:Lymphoid Tissue / Q000276:immunology; D008969:Molecular Sequence Data; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D014733:Vibrio / Q000502:physiology; D014735:Vibrio Infections / Q000276:immunology / Q000662:veterinary
D002402:Cathepsin D